HOXA9 expression is a common feature of human acute myelocytic leukemia (AML) and high level expression is correlated with poor prognosis. Moreover, HOXA9 overexpression immortalizes murine marrow progenitors which are arrested at a promyelocytic stage of differentiation when cultured, and causes leukemia in recipient mice following transplantation of HOXA9 expressing bone marrow. Our laboratory is now focused on developing treatment strategies based on blocking HOXA9 activity. Our recent data showed that protein kinase C (PKC) mediates phosphorylation of HOXA9 on S 204 and T205 at the N-terminus of the homeodomain. PKC phosphorylation of HOXA9 on S 204 abrogated the protein's DNA binding in vitro and the ability of endogenous HOXA9 to form cooperative DNA binding complexes with PBX. This HOXA9 phosphorylation was correlated with differentiation of myeloid cell lines and HOXA9-immortalized bone marrow cells. We hypothesize that this myeloid differentiation was in part due to PKC-mediated phosphorylation of HOXA9, which decreases the DNA binding of the homeoprotein. We have also recently determined the first set of downstream gene targets for HOXA9. We propose three aims: 1) to show a causal relationship between HOXA9 phosphorylation and myeloid differentiation, by elucidating the relationship between phosphorylation and HOXA9 pathological activity; 2) part I - determine which PKC isoform acts on HOXA9; part 2 - develop strategies to block HOXA9 transforming activity by: A) using RNAi to directly block HOXA9; B) using PKC stimulators or ser/thr phosphatase inhibitors to block HOXA9 activity in immortalized cell and leukemia models; and C) determine the X-ray crystal structure of HOXA9, to understand how phosphorylation blocks activity. This information will form the basis for future rationale drug design of HOXA9 inhibitors; and 3) to elucidate the direct molecular targets of the HOXA9 transcription factor with and without the MEIS1 and PBX cofactor proteins. The long range goal of this aim is to develop methods to block the biological activity of these downstream effector pathways. Taken together these studies should move us closer to achieving drug-based therapies for the treatment of acute myelogenous leukemias.